Our previous studies identified a region on chromosome 4q that was linked to measures of insulin action. A candidate gene in this region is FABP2 which encodes the human intestinal fatty acid binding protein (IFABP). We identified a polymorphism in this gene which results in an alanine (Ala54) to threonine (Thr54) substitution at amino acid 54 of IFABP. We found a significant association between the Thr54-encoding IFABP genotype (frequency= 0.29) and increased fasting lipid oxidation rates and insulin resistance, and have further shown that recombinant Thr54 protein has a higher affinity for long-chain fatty acids as compared to recombinant Ala54 protein. To investigate the structural differences between these proteins, we have collaborated with J. Hamilton to solve the strucure of the Ala54 protein by 3-D NMR, and are currently solving the structure of the Thr54 protein by the same method. To investigate physiologic consequences of the IFABP substitution, we analyzed fatty acid transport across cells expressing either Ala54 or Thr54 IFABP. We produced permanently transfected Caco- 2 cell lines expressing equivalent amounts of Ala54 and Thr54 IFABP and compared their ability to transport long-chain fatty acids from their apical to basolateral surfaces. We found that 3H lipid was transported at a faster rate across the Thr54-expressing cells as compared to the Ala54-expressing cells. The nature of the basolaterally secreted 3H-lipids components was also analyzed and we found that Thr54-expressing cells secreted a 4-6 fold greater amount of triglyceride to the basolateral media than Ala54 cells. We have further analyzed endogenous liver FABP expression in Caco-2 cells transfected with intestinal FABP, since native enterocytes express both of these FABP's, but express them in ratios that differentiate from the crypts to the villi. We have found that cells transfected with intestinal FABP express lower levels of endogenous liver FABP, when compared to nontransfected cells. This suggests that there is some sort of co-regulation between these two proteins. We are currently investigating the co-regulation in transfected cells.